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. 2005 Oct 1;19(19):2307-19.
doi: 10.1101/gad.1340605.

The Ubiquitin Ligase Rnf6 Regulates Local LIM Kinase 1 Levels in Axonal Growth Cones

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Free PMC article

The Ubiquitin Ligase Rnf6 Regulates Local LIM Kinase 1 Levels in Axonal Growth Cones

Baris Tursun et al. Genes Dev. .
Free PMC article

Erratum in

  • Genes Dev. 2005 Nov 1;19(21):2643

Abstract

LIM kinase 1 (LIMK1) controls important cellular functions such as morphogenesis, cell motility, tumor cell metastasis, development of neuronal projections, and growth cone actin dynamics. We have investigated the role of the RING finger protein Rnf6 during neuronal development and detected high Rnf6 protein levels in developing axonal projections of motor and DRG neurons during mouse embryogenesis as well as cultured hippocampal neurons. RNAi-mediated knock-down experiments in primary hippocampal neurons identified Rnf6 as a regulator of axon outgrowth. Consistent with a role in axonal growth, we found that Rnf6 binds to, polyubiquitinates, and targets LIMK1 for proteasomal degradation in growth cones of primary hippocampal neurons. Rnf6 is functionally linked to LIMK1 during the development of axons, as the changes in axon outgrowth induced by up- or down-regulation of Rnf6 levels can be restored by modulation of LIMK1 expression. Thus, these results assign a specific role for Rnf6 in the control of cellular LIMK1 concentrations and indicate a new function for the ubiquitin/proteasome system in regulating local growth cone actin dynamics.

Figures

Figure 1.
Figure 1.
Rnf6 and RLIM proteins are differentially expressed in E12.5-E13 mouse spinal cord. Unless otherwise indicated, transversal vibratome sections at the thoracic level of wild-type mice are shown. Immunohistochemical staining of E12.5-E13 mouse embryos using anti-Rnf6 (A) and anti-RLIM (B) polyclonal antibodies. High Rnf6 and RLIM expression is observed in similar regions of the developing ventral neural tube where motor- and inter-neurons reside and the floorplate (arrow). (C) Diffused Rnf6 staining in the developing mouse embryo indicating cytoplasmic expression. Higher magnification of the region boxed in A. (D) RLIM is highly expressed in nuclei of cells during mouse neural tube development. Higher magnification of the region boxed in B. Rnf6 staining in putative axonal projections of E12.5-E13 wild-type (E) and erbB3-/- (F) mouse embryos. The arrows indicate staining of axonal projections.
Figure 2.
Figure 2.
Rnf6 regulates axonal growth in primary hippocampal neurons. (A) Rnf6 is expressed in growth cones of primary hippocampal neurons. Neurons were cultured for 36 h and stained with Rnf6 antibodies (green) and antibodies directed against the axonal marker Tau (red). Note strong Rnf6 staining in the growth cone of the projection with the highest Tau levels, which will most likely develop into the axon. (Right panel) A phase-contrast image of the same neuron. Bar, 10 μm. (B) Treatment of primary hippocampal neuron cultures with Rnf6 siRNA decreases the level of Rnf6 protein. Western blot of protein extracts of Rnf6 siRNA and control siRNA (c-Rnf6 siRNA) transfected primary hippocampal neuron cultures probed with specific Rnf6 as well as with RLIM and β-tubulin antibodies as specificity and loading controls, respectively. (C) Down-regulation of Rnf6 in primary hippocampal neurons results in increased axon length. Primary neurons were cultured for 24 h prior to cotransfection of siRNAs with the empty pEGFP-C3 vector to visualize transfected cells. Twenty-four hours later, cells were processed for immunocytochemistry and stained with anti-neurofilament antibody. GFP signals (green, upper panels) and neurofilament staining (red, lower panels) of representative projections are shown. Bar, 10 μm. (D) Statistical analysis of axon lengths of Rnf6 siRNA-transfected neurons. The mean values of three independent transfections of control siRNA and Rnf6 siRNA are shown. The total number of neurons measured is indicated for each transfected siRNA. Neurofilament-positive projections were measured using analySIS Pro software. (**) P < 0.0001. Error bars indicate SEM. (E) Analysis of the axon lengths of primary hippocampal neurons transfected with constructs expressing GFP-Rnf6, GFP-Rnf6ΔRING, and GFP-C3. Neurofilament-positive projections were measured, using analySIS Pro software. Representative neuronal projections of transfected cells are shown. Bar, 10 μm. (F) Statistical analysis of axon lengths of neurons transfected with GFP fusion constructs. The mean values of three independent transfections are shown. The total number of neurons measured is indicated for each transfected construct. (**) P < 0.0001. Error bars indicate SEM.
Figure 3.
Figure 3.
Rnf6 associates with LIMK1 in cells. (A) Rnf6 and RLIM interact with LIMK1 in vitro. (Upper panel) 35S-labeled in vitro translated full-length mouse and rat LIMK1 (mLIMK1 and rLIMK1) proteins were tested for their ability to interact with GST-Rnf6 and GST-RLIM fusion proteins. (Lower panel) As input control, half of the reaction was run in parallel on a separate gel and proteins were visualized by Coomassie blue staining. (B) Overexpressed Rnf6 and RLIM interact with overexpressed LIMK1 in cells. Cell extracts of HeLa cells transfected with Myc- and HA-tagged constructs were immunoprecipitated with the indicated antibodies. To obtain higher protein levels, we used Rnf6 and RLIM constructs with deleted RING fingers. The Western Blot was probed with anti-HA antibodies. For negative controls, cells were cotransfected with HA-LIMK1 and the empty Myc vector pCS2-MT, which expresses six Myc epitops, or with the nonrelated Myc-Lhx3 expression plasmid. The arrow indicates the band expected for HA-LIMK1. (C) Interaction of endogenous Rnf6 and LIMK1 in primary hippocampal neuron cultures. Total cell extracts of primary hippocampal neurons, treated with the proteasome inhibitor MG132 for 6 h, were immunoprecipitated with either anti-LIMK1 mAb or nonspecific IgGs. The Western blot was probed with anti-Rnf6 or, as control, anti-LIMK1 antibodies. Twenty percent input is shown. The arrows indicate the bands expected for Rnf6 or LIMK1.
Figure 4.
Figure 4.
Rnf6 polyubiquitinates and targets LIMK1 for proteasomal degradation. (A) Rnf6 is a RING-finger-dependent ubiquitin ligase. In vitro 35S-labeled full-length Rnf6 protein or the RING-finger-deletion mutant Rnf6ΔRING (Δ589-667) protein was incubated with or without the ubiquitin-conjugating (E2) protein UbcH5. The arrow indicates the position of the nonubiquitinated Rnf6 proteins. Polyubiquitinated proteins are indicated by three asterisks (***). Note that in contrast to full-length Rnf6, the RING-finger-deletion mutant cannot autoubiquinate. (B) Bacterially expressed Rnf6 and RLIM mediate ubiquitination of 35S-labeled LIMK1 protein in vitro. The arrow indicates nonubiquitinated LIMK1 protein. Polyubiquitinated proteins are indicated by three asterisks (***). (C) Rnf6 polyubiquitinates LIMK1, generating K48 isopeptide linkages. Wild-type (wt) ubiquitin, ubiquitin lacking all lysine residues (K0), or an ubiquitin containing only one lysine at position 48 (K48only) was used for the in vitro ubiquitination assays. The arrow indicates nonubiquitinated LIMK1 protein. Polyubiquitinated proteins are indicated by three asterisks (***). Note that incubation with the K0 ubiquitin mutant protein results in little LIMK1 ubiquitination, whereas ubiquitination reactions using the K48only ubiquitin mutant protein were almost as efficient as those using wild-type ubiquitin. (D) Rnf6 targets LIMK1 protein for degradation. Western blots of total protein extracts of HEK293T cells cotransfected with Myc-LIMK1 and untagged RLIM, Rnf6, or the deletion mutant Rnf6ΔRING constructs were probed with anti-Myc antibodies. (E) Rnf6 decreases the half-life of LIMK1. GST-LIMK1 was expressed in Cos7 with or without Rnf6, and the proteins were labeled with 35S-methionine for 1 h, followed by washings and chasing in normal medium for time periods up to 7 h in the presence or absence of MG132. At each time point, GST-LIMK1 was pulled-down and subjected to Western blotting followed by exposure to PhosphorImager. (F) Rnf6 targets endogenous LIMK1 for degradation. HeLa cells were transfected with the dominant-negative Myc-Rnf6ΔRING expression plasmid. Cells were stained with anti-Myc (green) and anti-LIMK1 (red) antibodies. Note that endogenous LIMK1 levels (red) are greatly increased in cells transfected with Myc-Rnf6ΔRING (green).
Figure 5.
Figure 5.
Rnf6 expression partially colocalizes with LIMK1 in neuronal projections. (A) LIMK1 and Rnf6 proteins display an overlapping expression pattern during mouse nervous system development. Vibratome sections of E12.5-E13.5 erbB3+/- and erbB3-/- mice were stained with anti-Rnf6 polyclonal and anti-LIMK1 monoclonal antibodies. Arrows indicate stained axonal projections. (B) Rnf6 and LIMK1 partially colocalize in peri-nuclear punctae of hippocampal neurons. Mouse primary hippocampal neurons were cultured for 4 d and stained with anti-Rnf6 (red) and rat anti-LIMK1 antibodies (green). The yellow color indicates colocalization of both proteins (merge). Bar, 10 μm. (C) Rnf6 and LIMK1 partially colocalize in projections of hippocampal neurons. Mouse primary hippocampal neurons were cultured for 4 d and stained with anti-Rnf6 (red) and LIMK1 antibodies (green). Bottom panels show higher magnification of the boxed regions in the top panels. Arrows indicate punctuate regions of colocalization. Bar, 10 μm. (D) Colocalization of Rnf6 and LIMK1 in growth cones is restricted to a small interface region of the two proteins. Primary hippocampal neurons cultured for 2 d were stained with anti-Rnf6 (red) and anti-LIMK1 antibodies (green). Neurofilament-positive projections are shown. Bar, 4 μm. Images were captured by confocal microscopy using a 63× objective. Enlargement of the boxed region is shown in the lower panel. Note the small area of colocalization as indicated by arrows.
Figure 6.
Figure 6.
Proteasomal regulation of LIMK1 in growth cones. (A) Proteasomal regulation of LIMK1 levels in cultures of dissociated hippocampal neurons. Western blot analysis of total protein extracts of primary hippocampal neuron cultures treated with the proteasome inhibitor MG132 for 1, 3, and 6 h, probed with anti-LIMK1 and β-tubulin antibodies. (B) LIMK1 concentrations at the growth cones are regulated by the proteasome. Primary hippocampal neurons cultured for 2 d were treated for 1, 3, and 6 h with MG132, followed by staining with anti-LIMK1 antibodies (green). Only neurofilament-positive projections are shown. The time-dependent increase in LIMK1 concentrations was so high that the photomultiplier of the confocal microscope was set to a 50% lower level for cells treated for 3 and 6 h with MG132. Representative cells from three independent experiments are shown. (C) Relative LIMK1 levels in axonal growth cones of hippocampal neurons after 6 h of MG132 treatment as measured by fluorescence intensity of same-sized growth cone areas in three independent sets of experiments. Relative fluorescence intensities at 0 h of MG132 treatment were measured and normalized as 1. The total number of growth cones measured is indicated. (**) P < 0.0001. (D) The mouse UbcH5 ortholog is expressed in growth cones. Primary hippocampal neurons cultured for 2 d were stained with anti-UbcH5 antibodies (red). A neurofilament-positive projection is shown. (Right panel) A phase-contrast image of the same neuron. (E) Treatment with proteasome inhibitor results in colocalization of Rnf6 and LIMK1 over large parts of the growth cone. Primary hippocampal neuron cultured for 2 d and treated with MG132 for 3 h were stained for Rnf6 (red) and LIMK1 (green). Only neurofilament-positive projections are shown. Bar, 4 μm. Images were captured by confocal microscopy using a 63× objective. Enlargement of the boxed region is shown in the right panel. Note that colocalization of Rnf6 and LIMK1 occurs over large parts of the growth cone. Similar results were obtained in five independent experiments.
Figure 7.
Figure 7.
Rnf6 regulates LIMK1 levels in axonal growth cones. (A) Western blot of total protein extracts of primary hippocampal neuron cultures transfected with Rnf6 siRNA and control siRNA (c-Rnf6 siRNA) was probed with Rnf6 antibodies. The same blot was reprobed with LIMK1 antibodies and, as a loading control, with RLIM antibodies. Note that in cells transfected with Rnf6 siRNA, Rnf6 levels are decreased while LIMK1 levels are increased when compared with control-transfected cell extracts. Transfection efficiencies were ∼70%. (B) Representative growth cones of primary hippocampal neurons cotransfected with control siRNA or Rnf6 siRNA, together with the GFP-C3 expression vector to visualize transfected neurons. Cells were stained with Rnf6 (red) and LIMK1 (blue) antibodies. Bar, 6 μm. Note that in Rnf6 siRNA-transfected cells Rnf6 levels decrease, whereas LIMK1 levels increase. (C) Overexpressed GFP-Rnf6 does not colocalize with endogenous LIMK1 in growth cones. Primary hippocampal neurons were transfected with GFP-Rnf6, GFP-Rnf6ΔRING, and, as a control, GFP-C3 expression plasmids. GFP is shown in green, LIMK1 in red. Only neurofilament-positive projections are shown. Images were captured by confocal microscopy using a 63× objective. Enlargement of the boxed regions is shown to the right. Bar, 4 μm. Note that colocalization of GFP and GFP-Rnf6ΔRING with LIMK1 occurs over large parts of the growth cone, whereas GFP-Rnf6 colocalizes with endogenous LIMK1 only upon inhibition of the proteasome by MG132.
Figure 8.
Figure 8.
Rnf6 is functionally linked with LIMK1 during axonal growth. (A) Western blot of total protein extracts of primary hippocampal neuron cultures transfected with LIMK1 siRNA or control siRNA (c-LIMK1 siRNA) was probed with anti-LIMK1 mAb and anti-β-tubulin antibodies as loading controls. Note that in cells transfected with LIMK1 siRNA, the protein levels of LIMK1 are decreased in comparison with control-transfected cell extracts. Transfection efficiencies were ∼75%. (B) The increase in axon length mediated by Rnf6 siRNA is dependent on LIMK1. Dissociated primary hippocampal neurons were cultured for 24 h prior to cotransfection of siRNAs with the empty pEGFP-C3 vector to visualize transfected cells. Twenty-four hours later, cells were stained with anti-Tau antibody. GFP signals (green) and Tau staining (blue) of representative projections are shown. Bars, 10 μm. Note that the length of axonal projections of neurons cotransfected with both Rnf6 and LIMK1 siRNAs is similar to that of LIMK1 siRNA-transfected neurons. (C) Statistical analysis of axon lengths of neurons cotransfected with Rnf6 and LIMK1 siRNAs. Mean values of three independent experiments are shown. The total number of measured neurons is indicated. Tau-positive projections were measured, using analySIS Pro software. (**) P < 0.0001. Error bars indicate SEM. (D) Opposing roles of Rnf6 and LIMK1 for axon outgrowth. Primary neurons were cultured for 24 h prior to cotransfection of Myc or GFP expression constructs. Twenty-four hours later, cells were processed for immunocytochemistry and stained with anti-Tau and Myc-antibodies. GFP signals (green) and Tau and Myc stainings (blue and red, respectively) of representative projections are shown. Bars, 10 μm. Note that the length of axonal projections of neurons coexpressing Rnf6 and LIMK1 is similar to control-transfected neurons. (E) Statistical analysis of the lengths of primary hippocampal neurons cotransfected with Rnf6 and LIMK1 expression constructs. Mean values of three independent transfection experiments are shown. The total number of neurons measured is indicated for each transfection. Cells were stained with anti-Myc and anti-neurofilament antibodies. Tau-positive projections were measured using analySIS Pro software. (**) P < 0.0001. Error bars indicate SEM.

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